Retention time troubleshooting

[Ruben Ben]

Dear Forun

I am working with a HPLC method for a basic drug at 30 nm and mobil phase EDTA + TEA+ ACNO + water. Column previusly flushed 2o hs to eliminate cations.

Problemns is fluctuating Rrt Times ( To is constant). Variations of tr is random and happened before of 4 or 5 injeections.

I check for not leaks, enougth stabilization of column, new column and still it happened with sample and standard

RT is 7 minutes.

May be desetabilization between iniections ?. Any other idea or help

Thanks
Ruben

[bookoon]

Can you provide a little more details like the type/ make of column you are using, the exact composition of the mobile phase, column temperature etc.?

[Mark Tracy]

The retention time can be affected by 1) flow rate 2) mobile phase composition 3) temperature 4) stationary phase 5) sample

Since the t0 is constant, flow rate is probably not the problem. You can double-check by collecting the pressure as a second data channel, and look for unexpected shifts. This could be a bad check valve or occasional bubbles.

Stationary phase changes don't cause random shifts in tR

The mobile phase is the most likely problem. Not what you put in the bottle, but that it is not the same when it arrives at the column. One possibility is a leaky proportioning valve. Another is the autosampler loop is filled with something incompatible.

You didn't say if the samples were all the same or not, but sometimes the sample pH or solvent can perturb the retention time. Usually this will be accompanied by changes in the peak shape.

[Ruben Ben]

Dear All

I could solve the retention time problems with a stabilization overnigth, but believe or not, the real problem now is that areas of standard and also samples increase during the run. For example a same vial of standard give areas 100-101-100 and when the same vial is runing after others vials ( 4 to 6 injections) of standards and samples areas are for example, 130-129-130 and foloow increasing.

Mobil Phase: 500 ml EDTA 0,9 % + 500 ml of acetonitrle + 3 ml TEApH 8
Flow 1 ml x min
Column: Similar Zorbax Extend C18 250 x 4.6 ( Jupiter C18)
T Col: 30 C
Detection: 300 nm
Drug: amine derivade

I check diluting concentratio 4 to 1 rate with the same problem. No other peaks appear during cromatogram
The equipment does not have reproducibility problems with other column, phase mobil and drug.

May be a packaging column problem

Thanks

[mskishore13]

what is your sampling media? is it highly volatile solvent?

[Celiagg]

Hello Rubén

1.Frist at all, I would meassure the flow (better, with a glass flask of 10mL and a chromometer). Check that it is constant several times, and if you work in gradient way, with each of two phases separately, ( if each solvent goes by different valve...).

2.Once you have descarte the pump, I'll try to degass solvents properly with Helio bubles during 1/2 hour at least, to guarantee that are no bubbles of air the reason of migrating retention times.

3.After this, I would try to fix temperature. Some analyte's tr change a lot with temperatures changes of 3 ºC or less...

4. I would be sure of stability of the sample in the solvent mix that you use to dissolve it to inject . I'd make the same synthetic sample several times and after, I'd inject them with 1 hour between them: I mean, first fresh , second that is an hour from preparation...and so. Sorry, my Englihs is too old...

If your analytes are reacting between them, the product may be more absortive in that lambda. So areas may change.

5. At the end, I would try to investigate how long is since someone put the lamp into the detector. It may be low of energy, and make fluctuations in light. emission.

6. Are you using fast HPLC columns? If they are not well packed, it may make problems. like double peaks and losts of stationary phase. But it is not probable ...

7. You must be sure that you mixture well th mobile phase before start .

And Thats all i can say...you can write to me at cegrunder@yahoo.es

Si quieres comunicarte en español, no hay problema...
Regards

[XiZ]

Hi All!!

I'm having the same problem with the usp dissolution method for amoxicillin tablets (pH 5 potassium phosphate Buffer: ACN - 97,5: 2,5).
After 2 or 3 injections the RT jumps 3 or 4 minutes (+- 5 to +- 9).

In this case I'm thinking it's because we have too much water in the mobile fase making silanol groups lie down. Now the question: Is this right?

[tom jupille]

we have too much water in the mobile fase making silanol groups lie down. Now the question: Is this right?

No.

A much more likely source of problems is phosphate "buffer" at pH 5. You are outside the buffering range of phosphate at that point.

[Bruce Hamilton]

Hi All!!

I'm having the same problem with the usp dissolution method for amoxicillin tablets (pH 5 potassium phosphate Buffer: ACN - 97,5: 2,5).
After 2 or 3 injections the RT jumps 3 or 4 minutes (+- 5 to +- 9).

In this case I'm thinking it's because we have too much water in the mobile fase making silanol groups lie down. Now the question: Is this right?

I don't have latest USP, but I assume it's the same method as in 27
You presumably are following the method exactly, and the phosphate buffer is prepared as specified, and the samples are fresh ( < 6 hours ) and filtered?. The conditions should as specified - especially the 40C column temperature and different 2 cm guard column ( L2 ) for the main ( L1) column?.

Assuming you are following the method, my suspicion would be be that your sample injections are not dilute enough and are affecting the mobile phase. Do you see the jump with the standards, or only with dissolution samples. If it's with the standards, you need to look closely at your mobile phase preparation to ensure you are following the preparation method exactly, and giving the instrument plent of equilibrium time, including flushing the injector .

Bruce Hamilton

[XiZ]

Hi All!!

I'm having the same problem with the usp dissolution method for amoxicillin tablets (pH 5 potassium phosphate Buffer: ACN - 97,5: 2,5).
After 2 or 3 injections the RT jumps 3 or 4 minutes (+- 5 to +- 9).

In this case I'm thinking it's because we have too much water in the mobile fase making silanol groups lie down. Now the question: Is this right?

I don't have latest USP, but I assume it's the same method as in 27
You presumably are following the method exactly, and the phosphate buffer is prepared as specified, and the samples are fresh ( < 6 hours ) and filtered?. The conditions should as specified - especially the 40C column temperature and different 2 cm guard column ( L2 ) for the main ( L1) column?.

Assuming you are following the method, my suspicion would be be that your sample injections are not dilute enough and are affecting the mobile phase. Do you see the jump with the standards, or only with dissolution samples. If it's with the standards, you need to look closely at your mobile phase preparation to ensure you are following the preparation method exactly, and giving the instrument plent of equilibrium time, including flushing the injector .

Bruce Hamilton

Hi Bruce!!
Thx for your response.
We are using this method: http://www.pharmacopeia.cn/v29240/usp29 ... m4170.html (note that you can find all usp monographs on this site), but we are using a C18 25, with C18 guard column (a C8 guard column dídn't make any difference on the chromatogram). All the other specifications are exactly according to the method. Sometimes we use NaOH 50% (w/v) to adjust the pH. The samples are fresh and filtrated with some millex filters and Oven = 40C.

This RT jump occurs on both samples and standards, and the instrument are quite stable with mobile fase (10~20 mins with mobile Fase). I didn't mentioned that RT is very instable along the 93 runs of the dissolution samples, sometimes seems that it is dancing along the chromatogram.

(Sorry for my very poor english.)

At this momment I almost agree with the Tom's Idea about the buffering range (pH 5). What do you think? Any help would be very welcome.

[Uwe Neue]

Your problem will go away if you use the same column that was used to develop the method. The column that is specified is a 30 cm 3.9 mm column. This is a unique code for a microBondapak C18 column from Waters. The method was developed on this column, and the column is still available today. If you try another column, you will get random results.

[Celiagg]

Heloo All! Hello Rubén again!!


I've been thinking in your problem.

There is something I have not thought before. ¿How do you prepare the buffer? It sounds fool, but are you adjusting pH of the hole mixture (EDTA + TEA+ ACNO + water) at the end, after have made the mixture?.

pH determines molecules % of protonation. So the way in what the molecule interactuates with your stationary phase depends on pH , and its retention time too.

If rt woldn't change, but did not match with bibliography ones, I was thinking that the problem is column, but the problen is that it is not constan, so i think the cause is mobile phase.

REgars,

please, in spanish i could jhelp you better. Write to me to cegrunder@yahoo.es

[Mark Tracy]

It is quite OK to adjust the pH of the mobile phase after mixing the organic solvent. It is also OK to adjust the pH before mixing the organic solvent. But you must always do it the same way, and make sure that this is written in your SOP. This is discussed in the FAQ.

[Ruben Ben]

Dears

Thanks for everybody.

The mobile phase is run in 1 via ( isocratic) and I check the pH after mixing the organic solvent. I will try to change the column
Celia: Te escribire a tu e mail en español. Gracias